Bone fractures are a common traumatic injury. Approximately 8-10 million bone fractures are reported annually in the United States with more than 1 million of these requiring hospitalization. The estimated annual costs of treating these fractures exceeds 20 billion dollars. While this is already significant, these numbers are expected to increase due to the aging of the general population. Further, among military personnel, bone fractures are common training injuries. Bone fractures, typically located in the arms and legs, are also common battle wounds. Aside from traumatic injury, bone fractures also can be caused by disease. Osteoporosis is caused by a reduction in bone mineral density in mature bone and results in fractures after minimal trauma. The disease is widespread and has a tremendous economic impact. The most common fractures occur in the vertebrae, distal radius and hip. An estimated one-third of the female population over age 65 will have vertebral fractures, caused in part by osteoporosis. Moreover, hip fractures are likely to occur in about one in every three woman and one in every six men by extreme old age.
Fracture healing is a complex tissue regeneration process that involves cell migration, proliferation, apoptosis, and differentiation in response to growth factors, cytokines, other signaling molecules, and to the mechanical environment. The temporal order and magnitude of each cellular process must be controlled for optimal regeneration. The normal events of fracture healing are described below as occurring in 4 phases. In the initial phase, hematoma formation and localized tissue hypoxia are the initial cellular and molecular events of fracture healing. The second phase, called the early stage, is characterized by inflammation followed by rapid accumulation of cells at the fracture site. The presence of macrophages and neutrophils at the fracture site during inflammation precedes the rapid migration and proliferation of mesenchymal cells at the fracture site. In the third, regenerative phase, endochondral ossification creates the new bone which bridges the fracture. At this point, the fracture callus has a well-defined morphology. Intramembraneous ossification creates buttresses of periosteal bone at the callus periphery. Mesenchymal cells within the callus begin to differentiate into chondrocytes at the interface of the periosteal bone buttress. Each new chondrocyte develops as would be expected with matrix deposition followed by matrix calcification to produce calcified cartilage and then apoptosis. Channels are formed into the calcified cartilage starting at the periosteal bone buttresses. Osteoblasts migrate or differentiate on the surface of the calcified cartilage within these channels and begin depositing new bone. As chondrocyte differentiation proceeds from the periphery to the center of the callus (fracture site), channel formation, osteoblast differentiation, and new bone formation follows until the soft callus has been replaced with woven (immature) bone. Angiogenesis during the regenerative phase is essential. The immature woven bone created during the regenerative phase is mechanically unsuited for normal weight-bearing. To compensate for the decreased mechanical properties of the woven bone, the fracture callus has a significantly larger diameter which provides for greater structural mechanical properties. In the final, remodeling phase, fracture callus diameter diminishes until the bone obtains its normal dimensions while maintaining the bones overall mechanical properties by enhancing material mechanical properties. This is accomplished by replacing the mechanically poor, woven bone with mechanically strong, lamellar (mature) bone. In successive rounds, osteoclasts resorb the woven bone and osteoblasts replace it with lamellar bone. Molecular mechanisms governing osteoclast formation and function occurs through the RANKL-RANK pathway and this pathway is activated during fracture healing.
Fractures are generally treated conservatively by closed reduction of the fracture and immobilization (casting) of the affected bone. In such cases, the bone heals through the endochondral ossification pathway described above. Adequate nutrition to include vitamin C, vitamin D, and calcium aids in healing. There has been no major advancement in the treatment of bone fractures since the mid 20th century when open reduction and internal fixation of fractures became commonplace. The promise of growth factor treatments to enhance fracture healing has not been realized yet.
Unfortunately, many fractures require surgical intervention to increase healing success and reduce the likelihood of complication. There is only one approved pharmacological enhancement for bone healing and that is treatment with recombinant bone morphogenetic protein, either BMP-2 or BMP-7 (OP-1). Use of these growth factors requires surgery and due to expense and unknown potential side effects caused by the use of supraphysiological levels of growth factors, BMPs are used as a last-resort to heal recalcitrant fractures. Typical patient care also involves the administration of antibiotics, a narcotic, an NSAID, a COX-2 inhibitor or other pain killers during the healing process.
NSAIDs inhibit cyclooxygenase, thereby inhibiting the conversion of arachidonic acid into prostaglandins (PGD2, PGE2, PGF2α, PGI2, TXA2). Arachidonic acid is also a precursor for the leukotrienes (LTB4, LTC4, LTD4, LTE4), lipoxins (LXA4, LXB4), and 5-hydroxyeicosatetraenoic acid (5-HETE). The enzyme 5-lipoxygenase (5-LO) converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HpETE). This is the first step in the metabolic pathway which yields 5-HETE, the leukotrienes (LTs), and the lipoxins. Leukotrienes are also pro-inflammatory with the ability to attract neutrophils and cause capillary permeability. The arachidonic acid metabolic pathway is summarized in FIG. 1.
Lipoxygenases are nonheme iron-containing enzymes found in plants and animals that catalyze the oxygenation of certain polyunsaturated fatty acids, such as lipids and lipoproteins. Several lipoxygenase enzymes are known, each having a characteristic oxidation action. Mammalian lipoxygenases are named by the position in arachidonic acid that is oxygenated. For example, the enzyme 5-lipoxygenase converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HpETE), while the enzyme 12-lipoxygenas converts arachidonic acid to 12-HpETE. The activity of 5-lipoxygenase requires a co-factor commonly called FLAP (five lipoxygenase activating protein). Leukotriene synthesis is reduced by drugs that inhibit FLAP (MK866) or mice lacking FLAP.
WO 95/30419 discloses 5-LO inhibitors reduce osteoclast activity. The suppression of osteoclast activity inhibits bone resorption and reduces bone loss in human pathological conditions. Bone resorption is an integral part of fracture healing because it is necessary to remodel the newly formed bone into stronger, more mature bone. The inhibition of bone resorption would be expected to impair the later stages of normal fracture healing. Koivukangas et al., Long-term administration of clodronate does not prevent fracture healing in rats. Clinical Orthopaedics and Related Research 408: 268-278 (2003) and Peter et al. Effect of alendronate on fracture healing and bone remodeling in dogs. Journal of Orthopaedic Research 14: 74-79 (1996) disclose the effects of bisphosphonate therapy on fracture healing. The data show that bisphosphonate therapy which impairs osteoclast activity and bone remodeling does not inhibit the initial stages of fracture repair but does impair the later bone remodeling stage. The bisphosphonate effect on fracture healing reveals itself as persistence of a large fracture callus that contains mechanically immature, woven bone rather than mechanically mature, lamellar bone.
WO 03/066048 discloses that 12/15-lipoxygenase inhibitors can be used to prevent bone loss or increase bone mass. The publication describes data showing that bone mineral density is preserved in transgenic mouse that overexpress IL-4 and that were treated with a 15-LO inhibitor. The publication does not disclose that 15-LO inhibitors can aid fracture healing or the treatment of non-unions.
Traianedes, K., et al., 5-Lipoxygenase metabolites inhibit bone formation in vitro. Endocrinology, 139: 3178-3184 (1998) discloses the effects of LTB4,5-HETE, and LTD4 (all products of 5-LO function) on the differentiation of fetal rat calvaria (osteoblast) cells. The data show that 5-HETE and LTB4 reduce bone nodule formation and alkaline phosphatase activity in vitro but that LTD4 had no effect. The results from an in vitro organ culture model showed that LTB4 or 5-HETE treatment prevented a BMP2 induced increase in mouse calvaria thickness. The publication, however, does not disclose the use of any 5-LO inhibitors, nor does it disclose that 5-LO inhibition would lead to the same effect in cultured osteoblasts or in organ cultures. Similarly, Ren and Dziak, Effects of leukotrienes on osteoblast cell proliferation. Calcified Tissue International 49: 197-201 (1991) discloses that LTB4 treatment reduces proliferation of primary rat calvaria (osteoblast) cultures in vitro, but that LTB4 can promote proliferation of established osteoblast cell lines (Saos-2 and G292) in vitro at higher concentration (0.3-1 micromolar). Ren and Dziak also disclose that LTC4 had no effect on the proliferation of primary rat osteoblast cells or Saos-2 cells but did promote proliferation of G292 cells. Further, Ren and Dziak disclose that treatment of Saos-2 cells with a 5-LO inhibitor (AA-861) had no effect on Saos-2 cell proliferation. The publication indicates that 5-LO inhibition should have no effect on osteogenesis.
Thus, it is readily apparent that compositions and methods for accelerating or enhancing bone formation or fracture healing would be highly desirable.